Total internal reflection fluorescence microscopeA total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light.
FluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light.
Total internal reflectionIn physics, total internal reflection (TIR) is the phenomenon in which waves arriving at the interface (boundary) from one medium to another (e.g., from water to air) are not refracted into the second ("external") medium, but completely reflected back into the first ("internal") medium. It occurs when the second medium has a higher wave speed (i.e., lower refractive index) than the first, and the waves are incident at a sufficiently oblique angle on the interface.
Confocal microscopyConfocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object.
Oil immersionIn light microscopy, oil immersion is a technique used to increase the resolving power of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens. Without oil, light waves reflect off the slide specimen through the glass cover slip, through the air, and into the microscope lens (see the colored figure to the right).
Optical microscopeThe optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope.
Fluorescence microscopeA fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Microscope slideA microscope slide is a thin flat piece of glass, typically 75 by 26 mm (3 by 1 inches) and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then both are inserted together in the microscope for viewing. This arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders etc.
Super-resolution microscopySuper-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that use the Pendry Superlens and near field scanning optical microscopy) or on the far-field.
MicroscopeA microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope. There are many types of microscopes, and they may be grouped in different ways.
Infrared spectroscopyInfrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the interaction of infrared radiation with matter by absorption, emission, or reflection. It is used to study and identify chemical substances or functional groups in solid, liquid, or gaseous forms. It can be used to characterize new materials or identify and verify known and unknown samples. The method or technique of infrared spectroscopy is conducted with an instrument called an infrared spectrometer (or spectrophotometer) which produces an infrared spectrum.
Objective (optics)In optical engineering, an objective is an optical element that gathers light from an object being observed and focuses the light rays from it to produce a of the object. Objectives can be a single lens or mirror, or combinations of several optical elements. They are used in microscopes, binoculars, telescopes, cameras, slide projectors, CD players and many other optical instruments. Objectives are also called object lenses, object glasses, or objective glasses. The objective lens of a microscope is the one at the bottom near the sample.
Gaussian beamIn optics, a Gaussian beam is a beam of electromagnetic radiation with high monochromaticity whose amplitude envelope in the transverse plane is given by a Gaussian function; this also implies a Gaussian intensity (irradiance) profile. This fundamental (or TEM00) transverse Gaussian mode describes the intended output of most (but not all) lasers, as such a beam can be focused into the most concentrated spot. When such a beam is refocused by a lens, the transverse phase dependence is altered; this results in a different Gaussian beam.
Reflection (physics)Reflection is the change in direction of a wavefront at an interface between two different media so that the wavefront returns into the medium from which it originated. Common examples include the reflection of light, sound and water waves. The law of reflection says that for specular reflection (for example at a mirror) the angle at which the wave is incident on the surface equals the angle at which it is reflected. In acoustics, reflection causes echoes and is used in sonar. In geology, it is important in the study of seismic waves.
Fluorescence in the life sciencesFluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence (such as NADH, tryptophan or endogenous chlorophyll, phycoerythrin or green fluorescent protein). Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot.
Specular reflectionSpecular reflection, or regular reflection, is the mirror-like reflection of waves, such as light, from a surface. The law of reflection states that a reflected ray of light emerges from the reflecting surface at the same angle to the surface normal as the incident ray, but on the opposing side of the surface normal in the plane formed by the incident and reflected rays. This behavior was first described by Hero of Alexandria (AD c. 10–70). Later, Alhazen gave a complete statement of the law of reflection.
MicroscopyMicroscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy. Optical microscopy and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beams interacting with the specimen, and the collection of the scattered radiation or another signal in order to create an image.
Shot noiseShot noise or Poisson noise is a type of noise which can be modeled by a Poisson process. In electronics shot noise originates from the discrete nature of electric charge. Shot noise also occurs in photon counting in optical devices, where shot noise is associated with the particle nature of light. In a statistical experiment such as tossing a fair coin and counting the occurrences of heads and tails, the numbers of heads and tails after many throws will differ by only a tiny percentage, while after only a few throws outcomes with a significant excess of heads over tails or vice versa are common; if an experiment with a few throws is repeated over and over, the outcomes will fluctuate a lot.
Fluorescence spectroscopyFluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.
Terahertz time-domain spectroscopyIn physics, terahertz time-domain spectroscopy (THz-TDS) is a spectroscopic technique in which the properties of matter are probed with short pulses of terahertz radiation. The generation and detection scheme is sensitive to the sample's effect on both the amplitude and the phase of the terahertz radiation. Typically, an ultrashort pulsed laser is used in the terahertz pulse generation process. In the use of low-temperature grown GaAs as an antenna, the ultrashort pulse creates charge carriers that are accelerated to create the terahertz pulse.
